Systems and methods for continuous sorting of cells based on molecular adhesion

ABSTRACT

A microchannel for processing cells by compression of the cells including an inlet, ridges and an outlet. Each ridge including a compressive surface and a cell adhesion entity. The outlet configured to remove at least one of a first portion of the cells and a second portion of the cells from the microchannel. Each ridge oriented at an angle of from 25 degrees to 70 degrees relative to a center axis of the microchannel. The cell adhesion entity configured such that the first portion of the cells has a first adhesion property relative to the cell adhesion entity to follow a first trajectory through the microchannel. The cell adhesion entity further configured such that the second portion of the cells has a second adhesion property relative to the cell adhesion entity to follow a second trajectory through the microchannel. The first trajectory is different from the second trajectory.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation application of U.S. application Ser. No.16/348,520, filed on 9 May 2019, which application is a § 371 ofInternational Application No. PCT/US2017/060662, filed on 8 Nov. 2017,which International Application claims the benefit of U.S. ProvisionalApplication No. 62/419,534, filed on 9 Nov. 2016, each of which isincorporated herein by reference in its entirety as if fully set forthbelow.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant NumberCBET-0932510 awarded by the National Science Foundation and Grant Number1R1EB020977-01 awarded by the NIH. The Government has certain rights inthe invention.

THE NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT

Not Applicable

SEQUENCE LISTING

Not Applicable

STATEMENT REGARDING PRIOR DISCLOSURES BY THE INVENTOR OR A JOINTINVENTOR

Not Applicable

BACKGROUND OF THE DISCLOSURE 1. Field of the Invention

This invention relates generally to systems and methods for thecontinuous sorting of cells based on molecular adhesion, and moreparticularly to a microchannel for processing cells by compression ofthe cells.

2. Description of Related Art

Cell molecular interactions can regulate physiological processes, suchas cell homing, immune modulation, and cancer metastasis. Identifyingand isolating cells that express desired molecular surface markers canbe helpful to a variety of applications in the biological sciences, celltherapy, and medical diagnostics.

BRIEF SUMMARY OF THE INVENTION

In an exemplary embodiment of the present invention, a microchannel forprocessing cells by compression of the cells comprises an inletconfigured to deliver at least one of a first portion and a secondportion of the cells in the microchannel, multiple ridges, each of themultiple ridges comprising a compressive surface, and an outletconfigured to remove at least one of the first portion and the secondportion of the cells from the microchannel, wherein each of the multipleridges is oriented at an angle of from 25 degrees to 70 degrees relativeto a center axis of the microchannel, each of the multiple ridgescomprises a cell adhesion entity configured such that the first portionof the cells has a first adhesion property relative to the cell adhesionentity to follow a first trajectory through the microchannel and alsosuch that the second portion of the cells has a second adhesion propertyrelative to the cell adhesion entity to follow a second trajectorythrough the microchannel, and the first trajectory is different from thesecond trajectory.

The microchannel can further comprise a first wall and a second wall,wherein the first wall is substantially parallel to the second wall, andeach of the multiple ridges protrudes from the first wall in a directionnormal to the first wall and define a compression gap between thecompressive surface and the second wall.

The compressive surface of each of the multiple ridges can besubstantially parallel to each of the first wall and the second wall.

The compression gap can have a height of from 75% to 95% an averagediameter of the cells.

The microchannel can further comprise a flow space disposed between anadjacent pair of the multiple ridges along the center axis of themicrochannel, wherein the flow space has a width, along the center axis,from 50 micrometers to 500 micrometers. The flow space has the width,along the center axis, from 100 micrometers to 300 micrometers.

The microchannel can further comprise one or more sheath inlets, whereinthe inlet is a cell focusing inlet, and the one or more sheath inletssurround the cell focusing inlet.

The first adhesion property can be defined by a cell surface receptor ofthe cells, and the cell adhesion entity can temporarily be configured tobind to the cell surface receptor of the cells.

The first trajectory can be determined based on the first adhesionproperty, and the second trajectory can be determined based on thesecond adhesion property.

The microchannel can further comprise an additional outlet, wherein theoutlet is configured to remove the first portion from the microchannel,and the additional outlet is configured to remove the second portionfrom the microchannel.

The angle at which the multiple ridges relative to the center axis canvary along the center axis of the microchannel.

The multiple ridges can have a thickness that varies along the centeraxis of the microchannel.

The multiple ridges can have a thickness from 2 micrometers to 30micrometers.

The cell adhesion entity can be positioned on the compressive surface ofeach of the multiple ridges.

The cell adhesion entity can be positioned only on the compressivesurface of each of the multiple ridges.

The microchannel can comprise multiple types of cell adhesion entity,and different ones of the multiple types of the cell adhesion entity canbe positioned on the compressive surface of different ones of themultiple ridges.

The microchannel can comprise 5 to 100 compressive surfaces. Themicrochannel comprises 5 to 50 compressive surfaces. The microchannelcomprises 7 to 40 compressive surfaces.

Each of the multiple ridges can be oriented at an angle of from 30degrees to 60 degrees relative to a center axis of the microchannel.

In another exemplary embodiment of the present invention, a methodcomprises providing cells to a microchannel, the microchannel coated ina cell adhesion entity and comprising compressive surfaces and a firstoutlet, the compressive surfaces, formed by ridges oriented at an angleof from 25 degrees to 70 degrees measured with respect to a center axisof the microchannel, and defining compression gaps, each having a heightof from 75% to 95% of an average diameter of the cells, compressing thecells through the microchannel, wherein the compressing comprisesflowing the cells through the compression gaps which exposes the cellsto the cell adhesion entity, wherein the exposing causes a first portionof the cells having a first adhesion property relative to the celladhesion entity to follow a first trajectory through the microchanneland temporarily bind to the cell adhesion entity and a second portion ofthe cells having a second adhesion property relative to the celladhesion entity to follow a second trajectory through the microchannel,wherein the first adhesion property is different from the secondadhesion property, and wherein the first trajectory is different fromthe second trajectory, and collecting the first portion of the cells atthe first outlet.

The method can further comprise controlling flow resistance through thefirst outlet with a flow balancing region.

The method can further comprise collecting the second portion of thecells at a second outlet spaced away from the first outlet.

The microchannel can further comprise a first wall and a second wall,the first wall and the second wall being substantially planar to eachother, and wherein the compressive surfaces protrude in a directionnormal to the first wall and define the compression gaps between thecompressive surface and the second wall.

A flow space can be disposed between an adjacent pair of the compressivesurfaces along the center axis of the microchannel. A width of the flowspace, along the center axis, can be from 50 to 500 microns.

Providing the cells through the microchannel can further compriseproviding a sheath flow of a cell medium, to cause the cell medium toflow through the microchannel.

The first adhesion property can be defined by a cell surface receptor,and the cell adhesion entity can temporarily bind to the cell surfacereceptor.

The first trajectory can be determined based on the first adhesionproperty, and the second trajectory can be determined based on thesecond adhesion property.

The compressing can further comprise creating hydrodynamic circulationsof the cells within the compression gaps.

The angle at which the ridges are oriented can vary along the centeraxis of the microchannel.

The ridges can have a thickness that varies along the center axis of themicrochannel.

The cell adhesion entity can be positioned on the compressive surfaces.

The cell adhesion entity can be positioned only on the compressivesurfaces.

The cell adhesion entity can be positioned on the compressive surfaces,the first wall, and the second wall.

The microchannel can be coated in more than one type of cell adhesionentity, and different types of the cell adhesion entity can bepositioned on different ones of the compressive surfaces.

Providing the cells to the microchannel can comprise providing abiological fluid comprising the cells to the microchannel.

The biological fluid can be selected from the group consisting of blood,serum, and plasma.

Providing the cells to the microchannel can comprise providing a cellmedium comprising the cells to the microchannel.

The cell medium can be selected from the group consisting of a carbonsource, water, a salt, an amino-acid source, and a nitrogen-source.

Other exemplary embodiments of the present disclosure can include amethod comprising providing a plurality of cells to a microchannel, themicrochannel coated in at least one cell adhesion entity and comprisinga compressive surface and a first outlet, the compressive surfacedefining a compression gap, flowing the plurality of cells through themicrochannel, wherein the flowing comprises compressing the plurality ofcells underneath the compressive surface, and exposing the plurality ofcells to the at least one cell adhesion entity, wherein the exposingcauses a first portion of the cells having a first adhesion property totemporarily bind to the cell adhesion entity, and collecting the firstportion of cells at the first outlet, wherein the compression gap has aheight of from 75% to 95% an average diameter of the plurality of cells.

Other exemplary embodiments of the present disclosure can include amethod comprising flowing a cell medium through a microchannelcontaining at least one adhesion molecule, the cell medium comprising afirst cell having a first adhesion property and a second cell having asecond adhesion property, compressing the first and second cells as theyflow through the microchannel, wherein the compressing causes at leastone of the first and second cells to temporarily bind to the adhesionmolecule, and collecting one or both of the first and second cells at afirst outlet of the microchannel and collecting one or both of the firstand second cells at a second outlet of the microchannel.

Other exemplary embodiments of the present disclosure can include amethod comprising providing a cell medium to a microchannel, the cellmedium comprising a first cell having a first adhesion property and asecond cell having a second adhesion property, wherein the cell mediumis provided to the microchannel at a flow velocity of from 75 mm/s to300 mm/s, flowing the cell medium within the microchannel, themicrochannel defining a compression gap and coated in at least oneadhesion molecule, wherein the flowing comprises compressing the firstcell and the second cell as they pass through the compression gap,exposing the first cell and the second cell to the adhesion molecule,wherein the exposing causes the first and second cells to temporarilybind to the adhesion molecule, and collecting one or both of the firstand second cells at a first outlet of the microchannel and collectingone or both of the first and second cells at a second outlet of themicrochannel.

Other exemplary embodiments of the present disclosure can include adevice comprising an inlet for flowing a cell medium comprising aplurality of cells into the device at a flow velocity, a first planarwall and a second planar wall, the first planar wall having acompressive surface protruding normal to the first planar wall anddefining a compression gap between the second planar wall and thecompressive surface, a plurality of outlets for collecting sortedportions of the plurality of cells wherein the sorted portions shareadhesion properties, and at least one cell adhesion entity disposed onone or both of the first planar wall and the second planar wall, whereinthe compression gap has a height of from 75% to 95% an average diameterof the plurality of cells.

Other exemplary embodiments of the present disclosure can include asystem comprising a microchannel having a first planar wall and a secondplanar wall, the microchannel comprising a compressive surfaceprotruding outwardly from the first planar wall and defining acompression gap between the second planar wall and the compressivesurface, wherein the microchannel is coated with at least one adhesionmolecule, an inlet for flowing a cell medium into the microchannel at aflow velocity, the cell medium comprising a first cell having a firstadhesion property and a second cell having a second adhesion property, afirst outlet for collecting one or both of the first and second cellsand a second outlet for collecting one or both of the first and secondcells, wherein the plurality of cells are compressed as they flowthrough the compression gap, such that they temporarily bind with theadhesion molecule, and wherein the compression gap has a height of about80% to about 90% an average diameter of the plurality of cells.

In some exemplary embodiments, the microchannel of any of theabove-described systems, methods, and devices can further comprise afirst wall and a second wall, the first and second walls beingsubstantially planar to each other and the compressive surface isdisposed on the first and/or the second wall such that the compressivesurface protrudes normal to the first and/or second wall and defines thecompression gap between the compressive surface and a surface of thefirst and/or second wall. In some exemplary embodiments, themicrochannel of any of the above-described systems, methods, and devicescan further comprise at least one inlet. In some exemplary embodiments,the microchannel of any of the above-described systems, methods, anddevices can further comprise at least two outlets.

In any of the above-described systems, methods, and devices, at least aportion of the plurality of cells can undergo a compression due to thecompressive surface.

In any of the above-described systems, methods, and devices, themicrochannel can comprise from 1 to 7 compressive surfaces. In someexemplary embodiments, the microchannel comprises seven compressivesurfaces and at least a portion of the plurality of cells undergo sevencompressions. In of the above-described systems, methods, and devices,wherein the microchannel comprises more than one compressive surface,the microchannel can further comprise a flow space disposed betweenrespective compressive surfaces. In of the above-described systems,methods, and devices, the width of the flow space can be from 50 to 500microns. In some exemplary embodiments, the width of the flow space canbe from 100 to 300 microns. In some exemplary embodiments of any of theabove-described systems, methods, and devices, the compressive surfacecan comprise a ridge.

In some exemplary embodiments of any of the above-described systems,methods, and devices, the compressive surface(s) can be oriented at anangle of from 25 degrees to 70 degrees measured with respect to thecentral axis of the microchannel. In other embodiments, the compressivesurface(s) can be oriented at an angle of from 30 degrees to 60 degreesmeasured with respect to the central axis of the microchannel.

In some exemplary embodiments of any of the above-described systems,methods, and devices, the compression gap can have a height of from 80%to 90% the average cell diameter.

In some exemplary embodiments of any of the above-described systems,methods, and devices, the plurality of cells or cell medium can beprovided to the microchannel at a flow velocity of from 10 mm/s to 750mm/s. In other embodiments, the flow velocity can be about 75 mm/sec.

In some exemplary embodiments of any of the above-described systems,methods, and devices, a sheath flow can be provided to the plurality ofcells and/or cell medium, to cause the cells and/or cell medium to flowthrough the microchannel.

In some exemplary embodiments of any of the above-described systems,methods, and devices, the above-mentioned cells can comprise a cellsurface receptor and the cell adhesion entity temporarily binds to thecell surface receptor.

In some exemplary embodiments of any of the above-described systems,methods, and devices, one or more cells or portions of cells can havedifferent adhesion properties. In some exemplary embodiments of any ofthe above-described systems and methods cells having different adhesionproperties can follow different trajectories through the microchannel.

In some exemplary embodiments of any of the above-described systems,methods, and devices, from 40,000 to 100,000 cells can be sorted perminute based on one or more cell adhesion properties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a cross-sectional diagram of a cell sorting device, inaccordance with one or more embodiments of the present disclosure.

FIG. 1B is a schematic showing a three-outlet micro-channel for cellsorting having a plurality of diagonal ridges, in accordance with one ormore embodiments of the present disclosure.

FIG. 2 is a schematic showing a three-outlet microfluidic cell sortingdevice having a plurality of diagonal ridges, in accordance with one ormore embodiments of the present disclosure.

FIG. 3A is an adhesion-based sorting device, in accordance with one ormore embodiments of the present disclosure.

FIG. 3B shows the trajectories of Jurkat cells flowing through anadhesion-based sorting device, in accordance with one or moreembodiments of the present disclosure.

FIGS. 4A-4D are various graphical representations illustratingenrichment and fractionation of HL60 cells through adhesion, inaccordance with one or more embodiments of the present disclosure.

FIGS. 5A-5E shows various graphical representations illustratingtrajectories of HL60 cells with and without P selectin incubated deviceat different flow rates, in accordance with one or more embodiments ofthe present disclosure.

FIGS. 6A-6D show various graphical representations illustratingtrajectories of cells based on PSGL-1 expression, in accordance with oneor more embodiments of the present disclosure.

FIGS. 7A-7F show various graphical representations illustrating theeffects of gap size on cell enrichment, in accordance with one or moreembodiments of the present disclosure.

FIGS. 8A-8D show adhesion-based fractionation of cells, in accordancewith one or more embodiments of the present disclosure.

FIG. 9 shows results of a study analyzing activation of sorted cellsusing CD69 and CD11b staining, in accordance with one or moreembodiments of the present disclosure.

DETAILED DESCRIPTION

Although preferred exemplary embodiments of the disclosure are explainedin detail, it is to be understood that other exemplary embodiments arecontemplated. Accordingly, it is not intended that the disclosure islimited in its scope to the details of construction and arrangement ofcomponents set forth in the following description or illustrated in thedrawings. The disclosure is capable of other exemplary embodiments andof being practiced or carried out in various ways. Also, in describingthe preferred exemplary embodiments, specific terminology will beresorted to for the sake of clarity.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise.

Also, in describing the preferred exemplary embodiments, terminologywill be resorted to for the sake of clarity. It is intended that eachterm contemplates its broadest meaning as understood by those skilled inthe art and includes all technical equivalents which operate in asimilar manner to accomplish a similar purpose.

Ranges can be expressed herein as from “about” or “approximately” oneparticular value and/or to “about” or “approximately” another particularvalue. When such a range is expressed, another exemplary embodimentincludes from the one particular value and/or to the other particularvalue.

Using “comprising” or “including” or like terms means that at least thenamed compound, element, particle, or method step is present in thecomposition or article or method, but does not exclude the presence ofother compounds, materials, particles, method steps, even if the othersuch compounds, material, particles, method steps have the same functionas what is named.

Mention of one or more method steps does not preclude the presence ofadditional method steps or intervening method steps between those stepsexpressly identified. Similarly, it is also to be understood that themention of one or more components in a device or system does notpreclude the presence of additional components or intervening componentsbetween those components expressly identified.

Known methods for sorting cells based on adhesion, such as fluorescenceactivated cell sorting (FACS) or magnetic nanoparticle tagging for usein MACS, may not allow for fractionation into multiple outlets of finersensitivity to a molecule of interest. Other methods for what might beconsidered continuous sorting based on adhesion are incapable ofproviding high-throughput application. Additionally, other microfluidicapplications can rely on a process that results in cell capture insteadof continuous sorting where cells having certain affinity for theadhesive molecule attach to the device and can only be released by shearforces that can damage cells. Embodiments of the presently disclosedsystems and methods improve upon known devices in that they are capableof high-throughput separation of cells based on differences in molecularadhesion, allowing for fractionation of cells based on their tendency toattach to a surface, substrate, or another cell—a process mediated byinteractions between cell adhesion entities (e.g., adhesion molecules)providing specificity to a cell surface receptor. The describedembodiments can promote compression and relaxation of cells as cellsmove through a microchannel coupled with transient interaction betweencells and cell adhesion entities within the microchannel. Thecompressions can constrict the one or more cells as the cells passthrough the compressive space (e.g., a compression gap) during which thecells can transiently interact with the cell adhesion entity disposedupon the interfaces of the microchannel. As cells interact with the celladhesion entity, net forces can be altered to redirect the flowing cellson a specific trajectory towards certain portions of the channel. Forinstance, cells with high expression of target molecule (influenced bythe types of cell adhesion entity) can be concentrated toward one sideof the microchannel for collection.

The compressive spaces (e.g., compression gaps) and relaxation spacescan be designed to promote an increase in surface area for transient ortemporary interaction between the cell adhesion molecule and the cellsurface-which can be substantially without the influence ofbiomechanical properties such as stiffness and viscoelasticity. Someexemplary embodiments of the pending disclosure can also feature theability to obtain high throughput and high flow rate sorting of cellsbased on adhesion properties.

Embodiments of the present disclosure can comprise several systems andmethods for sorting cells based on adhesion properties. An exemplarymethod of the present disclosure can include one or more of thefollowing: (1) providing a plurality of cells to a microchannel, themicrochannel coated in at least one adhesion molecule and comprising acompressive surface and a first outlet, the compressive surface defininga compression gap; (2) providing a cell medium to a microchannel, thecell medium comprising a first cell having a first adhesion property anda second cell having a second adhesion property, wherein the cell mediumis provided to the microchannel at a flow velocity of from 10 mm/secondto 100 mm/second; (3) flowing the plurality of cells through themicrochannel; 4) flowing a cell medium through a microchannel containingat least one adhesion molecule, the cell medium comprising a first cellhaving a first adhesion property and a second cell having a secondadhesion property; (5) compressing the plurality of cells underneath thecompressive surface; (6) compressing the first and second cells as theyflow through the microchannel, wherein the compressing causes at leastone of the first and second cells to temporarily bind to the adhesionmolecule; (7) exposing the plurality of cells to the at least oneadhesion molecule, wherein the exposing causes a first portion of thecells having a first adhesion property to temporarily bind to theadhesion molecule; (8) collecting the first portion of cells at thefirst outlet; and (9) collecting one or both of the first and secondcells at a first outlet of the microchannel and collecting one or bothof the first and second cells at a second outlet of the microchannel.

In some exemplary embodiments, a method can comprise: providing aplurality of cells to a microchannel, the microchannel coated in atleast one adhesion molecule and comprising a compressive surface and afirst outlet, the compressive surface defining a compression gap;flowing the plurality of cells through the microchannel, wherein theflowing comprises compressing the plurality of cells underneath thecompressive surface; and exposing the plurality of cells to the at leastone adhesion molecule, wherein the exposing causes a first portion ofthe cells having a first adhesion property to temporarily bind to theadhesion molecule; and collecting the first portion of cells at thefirst outlet.

In some exemplary embodiments, a method can comprise: flowing a cellmedium through a microchannel containing at least one adhesion molecule,the cell medium comprising a first cell having a first adhesion propertyand a second cell having a second adhesion property; compressing thefirst and second cells as they flow through the microchannel, whereinthe compressing causes at least one of the first and second cells totemporarily bind to the adhesion molecule; and collecting one or both ofthe first and second cells at a first outlet of the microchannel andcollecting one or both of the first and second cells at a second outletof the microchannel.

In some exemplary embodiments, a method can comprise: providing a cellmedium to a microchannel, the cell medium comprising a first cell havinga first adhesion property and a second cell having a second adhesionproperty, wherein the cell medium is provided to the microchannel at aflow velocity of from 10 mm/s to 300 mm/s; flowing the cell mediumwithin the microchannel, the microchannel defining a compression gap andcoated in at least one adhesion molecule, wherein the flowing comprises:compressing the first cell and the second cell as they pass through thecompression gap; exposing the first cell and the second cell to theadhesion molecule, wherein the exposing causes the first and secondcells to temporarily bind to the adhesion molecule; and collecting oneor both of the first and second cells at a first outlet of themicrochannel and collecting one or both of the first and second cells ata second outlet of the microchannel.

In some exemplary embodiments, a device capable of achieving any of theabove-described methods can comprise: an inlet for flowing a cell mediumcomprising a plurality of cells into the device at a flow rate; a firstplanar wall and a second planar wall, the first planar wall having acompressive surface protruding normal to the first planar wall anddefining a compression gap between the second planar wall and thecompressive surface; a plurality of outlets for collecting sortedportions of the plurality of cells wherein the sorted portions shareadhesion properties; and at least one adhesion molecule coating themicrochannel.

In some exemplary embodiments, a system capable of achieving any of theabove-described methods can comprise: a microchannel having a firstplanar wall and a second planar wall, the microchannel comprising acompressive surface protruding normal to the first planar wall anddefining a compression gap between the second planar wall and thecompressive surface, wherein the microchannel is coated with at leastone adhesion molecule; a sheath inlet for flowing a cell medium into themicrochannel at a flow velocity, the cell medium comprising a first cellhaving a first adhesion property and a second cell having a secondadhesion property; a first outlet for collecting one or both of thefirst and second cells and a second outlet for collecting one or both ofthe first and second cells; wherein the plurality of cells arecompressed as they flow through the compression gap, such that theytemporarily bind with the adhesion molecule.

Any of the above-mentioned systems or methods can be used for sortingbased on adhesion properties. Sorting based on adhesion properties ofcells can be achieved by flowing cells through a microchannel. In someexemplary embodiments, the microchannel can be part of a microfluidicdevice.

FIGS. 1A and 1B illustrate an exemplary microchannel 100 for use in anyof the above-described systems and methods. As shown in FIG. 1A, themicrochannel 100 can comprise a top planar wall 110 and a bottom planarwall 120. The top planar wall 110 can comprise a plurality ofcompressive surfaces 130 protruding outwardly from the top planar wall110. The microchannel 100 can comprise one or more inlets 140 providedfor flowing a plurality of cells 180 and a plurality of particles 190into the microchannel 100.

While the first and second walls of the microchannel are described withrespect to FIGS. 1A and 1B as being planar, they need not be. Forinstance, they can be substantially planar. In other words, they can beslightly angled towards or away from each other such that they convergeor diverge across a length of the microchannel. In some exemplaryembodiments, they can converge or diverge more than slightly.

In some exemplary embodiments, and as illustrated at FIG. 1B, the one ormore inlets 140 may include a sheath flow inlet 145 a, 145 b fordelivering a sheath flow fluid into the microchannel 100 and a cellfocusing inlet 143. The microchannel 100 can comprise a plurality ofoutlets 150 for collecting portions of the plurality of cells 180.

The microchannel can comprise a plurality of compressive surfaces 130.In some exemplary embodiments, the plurality of compressive surfaces 130can comprise a plurality of ridges 133, as illustrated at FIG. 1B. Theplurality of ridges 133 may be any geometric shape that is substantiallyelongated across the micro-channel. In some exemplary embodiments, theplurality of ridges 133 may be diagonally-oriented with respect to acentral flow axis, as illustrated in FIG. 1B. The central flow axis canbe located proximate a central portion of the microchannel 100 and cancomprise an axis running parallel to a primary flow through themicrochannel 100. As illustrated at FIG. 1B, in some exemplaryembodiments, the plurality of ridges 133 can extend parallel to eachsubsequent ridge of the plurality of ridges. The plurality ofcompressive surfaces 130 may be straight, but need not be. For instance,the plurality of compressive surfaces 130 can be any shape, includingbut not limited to rectangular, cylindrical, trapezoidal, or triangular.Additionally, as will be understood by those skilled in the art, theplurality of compressive surfaces can comprise at least one ridge, butneed not all be ridges.

The plurality of compressive surfaces 130 may define a compression gap170 between a compressive surface 130 and a surface of an opposing wall120, as illustrated at FIG. 1A. For instance, in an embodiment whereinthe plurality of compressive surfaces 130 protrudes from the firstplanar wall 110, the plurality of compressive surfaces 130 may define acompression gap 170 between a compressive surface 130 and a surfaceacross from the compressive surface 130 on the second planar wall 120.As used herein, a surface may include the closest or nearest portion ofthe opposing wall, for example where the wall does not otherwise havecorresponding ridges or protrusions. In some exemplary embodiments, thesecond planar wall 120 can comprise a plurality of compressive surfaces130, and the opposing surface can be, for example, an opposingcompressive surface 130. The compression gap 170 can therefore bedefined as the space formed between a compressive surface 130 and asurface of the second wall 120, or the space between opposingcompressive surfaces on opposing walls. In some exemplary embodiments,the opposing ridges will be aligned with each other as well.

The size of the compression gap 170 can be increased or decreased asdesired, based on device design. In some exemplary embodiments, the sizeof the compression gap 170 can be defined in terms of the averagediameter of a cell. As will be understood, the diameter of the cell canbe defined as the largest distance between two points on a cell. In someexemplary embodiments, the height of the compression gap may be definedbased on a percentage of the average cell diameter. In some exemplaryembodiments, the compression gap can have a height that is from 75% to95% of the average cell size of the cells flowed through the cellsorting device (e.g., 75% to 90%, 80% to 90%, 85% to 95%, 75% to 85%,75% to 80%, 80% to 85%, or 80% to 95%)). In some exemplary embodiments,the compression gap can have a height that is 75% or greater of theaverage cell size of the cells flowed through the cell sorting device(e.g., 80% or greater, 82% or greater, 84% or greater, 86% or greater,88% or greater, 90% or greater, or 92% or greater). the compression gapcan have a height that is 95% or less of the average cell size of thecells flowed through the cell sorting device (e.g., 94% or less, 92% orless, 90% or less, 88% or less, 86% or less, 84% or less, 82% or less,80% or less, 78% or less, 76% or less). The average cell size can referto average of the largest cross-sectional dimension of the cells flowedthrough the sorting device, and can be calculated using. In someexemplary embodiments, the average cell diameter can be measured using avariety of tools now known or later discovered including but not limitedto optical microscopy, confocal microscopy, coulter counter, and flowcytometry.

As shown in FIGS. 1A and 1B, the plurality of compressive surfaces 130may be separated by a flow space 160. The flow space 160 can comprisethe width of a space or channel formed between a first compressivesurface of the plurality of compressive surfaces and a secondcompressive surface of the plurality of compressive surfaces. In someexemplary embodiments, the flow space 160 may be from 50 to 1000microns, from 50 to 750 microns, from 50 to 500 microns, from 50 to 400microns, from 50 to 350 microns, from 100 to 300 microns, from 100 to750 microns, from 100 to 500 microns, from 100 to 400 microns, from 100to 300 microns, from 100 to 250 microns, or from 125 to 250 microns. Theflow space 160 can be at least 50 microns, at least 100 microns, atleast 125 microns, at least 150 microns, at least 250 microns, or atleast 300 microns. The flow space 160 can be up to 5 microns, up to 3microns, up to 2 microns, up to 1 microns, up to 750 microns, or up to500 microns, 50 to 350 microns, from 100 to 300 microns, from 100 to 250microns, from 125 to 250 microns, or at least 300 microns.

The plurality of compressive surfaces 130 may comprise an angle (α), asillustrated at FIG. 1B. The plurality of compressive surfaces 130 can beinclined at an angle to create hydrodynamic circulations underneath thecompressive surfaces 130 and can be designed to compress and translatecells normal to the flow direction. The angle of the compressivesurfaces 130 can also affect the trajectories of cells. The angle mayvary depending on one or more parameters including, but not limited to,the types of cells flowed through the microchannel, the relaxation space160, and the flow velocity of the medium flowed through the microchannel100. As such, adjusting the angle may facilitate migration of cellsalong the compressive surfaces 130. For instance, adjusting the anglemay facilitate movement of dead or damaged cells to the sides of themicrochannel 100 to prevent clogging of the microchannel 100.

The angle may be increased or decreased, based on device design. Forinstance, in some exemplary embodiments, the angle can be from 20 to 75degrees, from 30 to 60 degrees, from 30 to 45 degrees, at least 20degrees, at least 30 degrees, at least 45 degrees, at least 60 degrees,or at least 75 degrees. The angle of each respective compressive surfacemay also be the same or different along a length of the microfluidicdevice. In instances where a compressive surface 130 is not linear, theangle can be measured based on a line that is a linear fit to thenon-linear ridge.

The number of compressive surfaces 130 in the microchannel 110 can beincreased or decreased as desired. In some exemplary embodiments, themicrochannel 110 can comprise 5 to 100 compressive surfaces 130. In someexemplary embodiments, the microchannel 110 can comprise at least threecompressive surfaces 130, at least four compressive surfaces 130, atleast five compressive surfaces 130, at least six compressive surfaces130, at least seven compressive surfaces 130, at least eight compressivesurfaces 130, at least nine compressive surfaces 130, or at least 10compressive surfaces 130. In some exemplary embodiments, themicrochannel 110 can comprise up to 100 compressive surfaces 130, up to75 compressive surfaces 130, up to 50 compressive surfaces 130, or up to40 compressive surfaces 130. In some exemplary embodiments, themicrochannel 110 can include 5 to 50 compressive surfaces 130, 7 to 40compressive surfaces 130, or 7 to 21 compressive surfaces 130. In someexemplary embodiments, the microchannel 110 can comprise 1fourcompressive surfaces 130.

The plurality of compressive surfaces 130 can be described by athickness. The thickness can be defined as the linear measurement of thecompressive surface in the direction of primary flow. The thickness canbe increased or decreased as desired. In some exemplary embodiments, thethickness can be from 2 to 30 microns, from 2 to 20 microns, from 2 to18 microns, from 2 to 16 microns, from 2 to 11 microns, from 2 to 9microns, from 5 to 30 microns, from 5 to 20 microns, from 5 to 18microns, from 5 to 16 microns, from 5 to 11 microns, from 5 to 9 micronsfrom 7 to 30 microns, from 7 to 20 microns, from 7 to 18 microns, from 7to 16 microns, from 7 to 11 microns, from 7 to 9 microns, from 9 to 20microns, from 9 to 11 microns, from 9 to 15 microns, from 9 to 17microns, from 9 to 30 microns, from 9 to 25 microns, from 10 to 20microns, from 15 to 20 microns, from 15 to 30 microns, from 20 to 30microns, from 22 to 28 microns, from 24 to 28 microns, from 18 to 21microns, from 16 to 22 microns, or from 8 to 11 microns. In someexemplary embodiments, the thickness can be at least 2, at least 5, atleast 7, at least 8, at least 9 microns, at least 11 microns, at least15 microns, at least 16 microns, at least 18 microns, at least 20microns, at least 22 microns, at least 24 microns, at least 25 microns,at least 27 microns and at least 30 microns.

The microchannel 100 can have one or more inlets 140. The one or moreinlets 140 may be located on a first side wall of microchannel 100. Insome exemplary embodiments, the microchannel 100 can have a cellfocusing inlet 143 and a sheath flow inlet 145 a, 145 b. In someexemplary embodiments, the cell focusing inlet 143 can accomplishinertial focusing. For instance, in some exemplary embodiments, the cellinlet can be located between a first sheath flow inlet 145 a and asecond sheath flow inlet 145 b, or can be surrounded by a first sheathflow inlet 145 aa. In some exemplary embodiments, the cell focusinginlet 143 can be downstream from one or more sheath flow inlets 145 a,145 b, or can be aligned with one or more sheath flow inlets 145 a, 145b. A sheath fluid can allow for hydrodynamic focusing of the cellmedium. The one or more sheath flow inlets 145 a, 145 b can be locatedproximate the cell flow inlet 147, or upstream of the cell flow inlet147. Focusing the cells in the inlet can comprise providing a sheathfluid to the sheath flow inlets 145 a, 145 b until the sheath fluidreaches laminar flow and then subsequently introducing the cell mediumcell medium through the cell inlet 147. The cells can be introduced intothe cell inlet 147 by injection, for example by syringe pumps.

The described microchannel 100 can be constructed in a variety of ways.In some exemplary embodiments, the microchannel can be made using areplica molding of polydimethylsiloxane (PDMS) on a permanent mold. Themold can be created by two-step photolithography patterning of aphotoresist on a 4-inch-diameter silicon wafer. After the removal ofPDMS from the mold, inlet and outlet holes can be punched in the sidewalls of the PDMS, and the PDMS can be subsequently bonded to a glasssubstrate to form the microfluidic channel. As will be understood bythose skilled in the art, the microchannel can be constructed of avariety of materials that may permit construction of compressivesurfaces in. Additionally, in some exemplary embodiments, the systemsand methods can include more than one microchannel to allow forincreased and simultaneous performance of the above-described methods.

The plurality of cells 180 can be flowed into the microchannel 100 at aflow velocity. The flow velocity of any of the systems and methodsdescribed previously can be increased or decreased as desired. As usedherein, the flow velocity can describe the velocity of the cell mediumat an inlet or at an outlet. The flow velocity can be from 3 to 1000mm/s, from 3 to 500 mm/s, from 3 to 250 mm/s, from 3 to 100 mm/s, from 3to 50 mm/s, from 3 to 25 mm/s, from 3 mm/s to 10 mm/s, from 10 mm/s to750 mm/s, from 10 mm/s to 500 mm/s, from 10 mm/s to 100 mm/s, from 10mm/s to 50 mm/s, from 10 mm/s to 25 mm/s. The flow velocity can be atleast 3 mm/s, at least 10 mm/s, at least 20 mm/s, at least t 50 mm/s, atleast 100 mm/s, at least 500 mm/s, or at least 750 mm/s. The flowvelocity can be 3 mm/s, 20 mm/s, 500 mm/s, 750 mm/s, or 1000 mm/s. Theflow velocity can also be adjusted as a function of the length of thechannel, and/or the size of the relaxation space, based on designpreferences. For instance, increasing the length of the channel canallow for a greater flow velocity. Increasing the velocity in similarlysized devices can result in increased pressure within the device. Byincreasing the length of the microchannel, the increased pressure can beaccounted for while permitting higher flow velocity. For instance,increasing the relaxation space can permit increasing the flow velocityas the greater space allows the cells a longer distance over which totravel and be subjected to secondary flow in the ridge channels. Assuch, increased relaxation space can permit an increased relaxation timeand positive lateral displacement for certain cells despite greater flowvelocity.

The microchannel can comprise a plurality of outlets 150 a, 150 b forcollecting sorted portions of the plurality of cells. FIG. 1Billustrates an exemplary three-outlet system where a first outlet 183 amay collect a first portion of cells having a first adhesion property, asecond outlet 183 b may collect a second portion of cells having asecond adhesion property, and a third outlet 183 c may collect thirdportion of cells having a third adhesion property By having amultiple-outlet system and modes of secondary flow (e.g. relaxationspaces between subsequent compressive spaces), the presently describedsystems and methods can achieve high-throughput sorting of cells basedon adhesion. In some exemplary embodiments, the microchannel 100 cancomprise at least two outlets, at least three outlets, at least fouroutlets, at least five outlets, at least seven outlets, or at least 10outlets.

When a cell medium is flowed through the microfluidic device, cells,depending on adhesion properties, may follow unique trajectories. Forinstance, as illustrated at FIG. 1B, as cells flow through themicrofluidic device and transiently interact with adhesion moleculescoating the compressive surfaces and walls of the channel, the netforces are altered to redirect the flowing cells toward one side of thechannel. Thus, for instance, cells with high expression of the targetmolecule are concentrated toward one side of the channel for collection.

The cell sorting device can comprise a plurality of outlets forcollecting sorted portions of the plurality of cells wherein the sortedportions share adhesion properties. For instance, in an embodiment,sorting cells based on adhesion, the cell sorting device can comprise atleast two outlets wherein one outlet collects cells with high expressionfor a target molecule and one outlet collects cells with low expressionof a target molecule. As discussed above, cells having differentadhesion properties may follow unique trajectories, e.g., cells maytravel through the device toward a particular outlet based on adhesionproperties. As will be understood, increasing the outlets can result inmore focused sorting with increased purity.

Any of the above-described outlets can include a well or chamber forpooling and/or pipetting them in the direction of a chamber or directlyto a chamber. In other embodiments, the outlets can be furtherintegrated with additional processing steps, as described below, throughan integrated chip or through a capillary. Additionally, after cells arecollected, any of the above-mentioned systems and methods can include anadditional step of analyzing the cells using any analysis tool now knownor later discovered including but not limited, flow cytometry,fluorescence microscopy, functional assays (e.g., apoptosis, cell cycle,viability, proliferation, angiogenesis), spectroscopy, immunoassays, andmicroplating. Additionally, in some exemplary embodiments, themicrochannel and cells can be analyzed with electrode counters andmicroscopy.

Any of the above-described systems and methods can include a variety ofcell adhesion entities. The at least one adhesion molecule can compriseone or more of selectins (e.g., E-selectins, P-selectins (PSGL1,L-selectins, etc.), integrins (e.g., an alpha integrin such as CD49a,CD49b, CD49c, CD49d, CD49e, CD49f, ITGA7, ITGA8, ITGA9, ITGA10, ITGA11,CD11D, CD103, CD11a, CD11b, CD51, ITGAW, CD11c, or a beta integrin suchas CD29, CD18, CD61, CD104, ITGB5, ITGB6, ITGB7, IT), cadherins (e.g.,E-cadherins, N-cadherins, P-cadherins, etc.), and immunoglobulin celladhesion molecules (e.g., SynCAMs, NCAMs, ICAM-1, VCAM-1, PECAM-1, L1,CHL1, MAG, nectins, CD2, CD48, SIGLEC family members such as CD22 andCD83, and CTX family members such as CTX, JAMs, BT-IgSF, CAR, VSIG, andESAM). In some exemplary embodiments, the cell adhesion entity caninclude proteins such as HER2. In some exemplary embodiments, the celladhesion entity can include antibodies (including antibody fragments)that can be on the surface of the device. In some exemplary embodiments,antibodies can be co-immobilized with one or more of other cell adhesionentities. In other embodiments, the cell adhesion entity can be one ormore engineered molecules now known or later discovered.

The cell adhesion entities can be disposed within or on themicrochannel. In some exemplary embodiments, the cell adhesion entitycan be disposed on one or more of the compressive surfaces, the firstplanar wall, and the second planar wall. In some exemplary embodiments,the entire microchannel can be coated in the cell adhesion entity. Inother embodiments only the compressive surfaces can be coated in thecell adhesion entity. In other embodiments only one or both of theplanar walls can be coated in the cell adhesion entity.

Any of the above-described systems and methods can include a variety ofcells and cell types. Additionally, the cell medium may contain one ormore different cell types and one or more different adhesion molecules.Any of the above-described systems and methods can achieve convectiveintracellular delivery of molecules into a variety of cell types. Thesecell types may include, but are not limited to cells of the reproductivesystem, e.g. oocytes, spermatozoa, leydig cells, embryonic stem cells,amniocytes, blastocysts, morulas, and zygotes; leukocytes, e.g.peripheral blood leukocytes, spleen leukocytes, lymph node leukocytes,hybridoma cells, T cells (cytotoxic/suppressor, helper, memory, naive,and primed), B cells (memory and naive), monocytes, macrophages,granulocytes (basophils, eosinophils, and neutrophils), natural killercells, natural suppressor cells, thymocytes, and dendritic cells; cellsof the hematopoietic system, e.g. hematopoietic stem cells (CD34+),proerythroblasts, normoblasts, promyelocytes, reticulocytes,erythrocytes, pre-erythrocytes, myeloblasts, erythroblasts,megakaryocytes, B cell progenitors, T cell progenitors, thymocytes,macrophages, mast cells, and thrombocytes; stromal cells, e.g.adipocytes, fibroblasts, adventitial reticular cells, endothelial cells,undifferentiated mesenchymal cells, epithelial cells including squamous,cuboid, columnar, squamous keratinized, and squamous non-keratinizedcells, and pericytes and also including limbal stem cells; cells of theskeleton and musculature, e.g. myocytes (heart, striated, and smooth),osteoblasts, osteoclasts, osteocytes, synoviocytes, chondroblasts,chondrocytes, endochondral fibroblasts, and perichonondrial fibroblasts;cells of the neural system, e.g. astrocytes (protoplasmic and fibrous),microglia, oligodendrocytes, and neurons; cells of the digestive tract,e.g. parietal, zymogenic, argentaffin cells of the duodenum,polypeptide-producing endocrine cells (APUD), islets of langerhans(alpha, beta, and delta), hepatocytes, and kupfer cells; cells of theskin, e.g. keratinocytes, langerhans, and melanocytes; cells of thepituitary and hypothalamus, e.g. somatotropic, mammotropic,gonadotropic, thyrotropic, corticotropin, and melanotropic cells; cellsof the adrenals and other endocrine glands, e.g. thyroid cells (C cellsand epithelial cells); adrenal cells; and tumor cells.

The cells may be Burkitt lymphoma cells, choriocarcinoma cells,adenocarcinoma cells, non-Hodgkin's B and T cell lymphoma cells,fibrosarcoma cells, neuroblastoma cells, plasmacytoma cells,rhabdomyosarcoma cells, carcinoma cells of the pharynx, renaladenocarcinoma, hepatoma cells, fibrosarcoma cells, myeloma cells,osteosarcoma cells, teratoma cells, teratomal keratinocytes, lungcarcinoma cells, colon adenocarcinoma cells, lung adenoma cells, renalcarcinoma cells, rectum adenocarcinoma cells, chronic myelogenousleukemia cells, ileocecal adenocarcinoma cells, hairy cell leukemiacells, acute myelogenous leukemia cells, colon carcinoma cells, cecumcarcinoma and adenocarcinoma cells, leukemia-cecum adenocarcinoma cells,pancreatic carcinoma, Wilm's tumor cells, prostate adenocarcinoma cells,renal leimyooblastoma cells, bladder carcinoma cells, plasmacytomacells, teratocarcinoma cells, breast carcinoma, epidermoid carcinoma ofthe cervix, ovarian teratocarcinoma, myeloma cells, T and B celllymphoma cells, amalanotic melanoma cells, cervical carcinoma cells,rhabdomyosarcoma, hepatoma, medullary Thyroid carcinoma cells, malignantmelanoma cells, glioblastoma cells, plasma cell leukemia, endometrialadenocarcinoma, squamous cell carcinoma, pancreatic adenocarcinoma,astrocytoma, gastric adenocarcinoma, pulmonary mucoepidermoid carcinomacells, myeloid leukemia cells, EBV-transformed B cells, renal celladenocarcinoma, acute leukemia, B cell plasmacytoma, acute lymphocyticleukemia, cutaneous T lymphoma, T cell leukemia, acute lymphoblasticleukemia, HIV+ T cells, medulloblastoma, B cells from sickle celldisease, acute monocytic leukemia, adrenocortical carcinoma, BowesMelanoma and hepatocellular carcinoma.

The plurality of cells in any of the above-described systems and methodsmay include any of the above cells or derivatives thereof. In someexemplary embodiments, the plurality of cells can include a mixture ofcell types. For instance, the cells can be part of a biological samplethat can comprise a cell, a tissue, a fluid (e.g., a biological fluid),a protein (e.g., antibody, enzyme, soluble protein, insoluble protein),a polynucleotide (e.g., RNA, DNA), a membrane preparation, and the like,that can optionally be further isolated and/or purified from its nativeor natural state. In some exemplary embodiments, the plurality of cellscan be in a “biological fluid” which can include any a fluid originatingfrom a biological organism. Exemplary biological fluids include, but arenot limited to, blood, serum, and plasma. A biological fluid may be inits natural state or in a modified state by the addition of componentssuch as reagents, or removal of one or more natural constituents (e.g.,blood plasma). A sample can be from any tissue or fluid from anorganism. In some exemplary embodiments, the sample can be a biopsy. Insome exemplary embodiments, the sample can comprise tissue from thebreast, digestive tract, lung, liver, kidney, brain, lip, mouth,esophagus, urinary bladder, prostate, vagina, and/or cervix. In someexemplary embodiments, the sample is from a tissue that is part of, orassociated with, the breast of the organism. In some exemplaryembodiments, the sample may be tissue from a neoplasm. A neoplasm mayinclude cancer. In some exemplary embodiments, the sample may becancerous tissue or from a tumor. In some exemplary embodiments, thesample may comprise tissue surrounding cancerous tissue or a tumor. Insome exemplary embodiments, the sample may comprise tissue surroundingor around the perimeter of cancerous tissue or a tumor that wassurgically excised. In some exemplary embodiments, the plurality ofcells being sorted comprises a mixed population of cell types. In someexemplary embodiments, the plurality of cells being sorted comprises amixed population of cancer cells and non-cancer cells. In some exemplaryembodiments, the plurality of cells being sorted comprises a mixedpopulation of metastatic cancer cells and non-metastatic cancer cells. Acell may be a normal or healthy cell. A cell may be a neoplasatic cell.A cell may be a cancer cell. Cancer may include a carcinoma, an adenoma,a melanoma, a sarcoma, a lymphoma, a myeloid leukemia, a lymphaticleukemia, a blastoma, a glioma, an astrocytoma, a mesothelioma, or agerm cell tumor.

Additionally, any of the above-described systems and methods can includecells or cell flow medium that include fluorescent tags, dyes,antibodies,

While the presently described systems and methods are described in termsof biological cells, it is understood that these presently disclosedsystems and methods can be achieved using a variety of materials otherthan biological cells, in some exemplary embodiments, theabove-described systems and methods can be achieved with a variety ofparticles, including nanoparticles, hydrogels, capsules, bacteria,viruses.

Additionally, any of the above-described systems and methods can includeany of the above-described cells suspended in a fluid, such as a cellmedium. The cell medium can be any liquid in which a plurality of cellscan be suspended and can include additional substances including one ormore of a carbon source (e.g., glucose) water, various salts, a sourceof amino acids and nitrogen (e.g., beef, yeast extract). Additionally,the medium may include other nutrients such as plant count agar,nutrient agar, trypticase soy agar, or a combination thereof.

Any of the above-described systems and methods can allow forhigh-throughput sorting. For instance, any of the above-describedsystems and methods can further comprise sorting from 10,000 to 500,000cells/min, from 10,000 to 450,000 cells/min, from 10,000 to 400,000cells/min, from 10,000 to 350,000 cells/min, from 10,000 to 300,000cells/min, from 10,000 to 250,000 cells/min, from 10,000 to 200,000cells/min, from 10,000 to 150,000 cells/min, from 10,000 to 100,000cells/min, from 10,000 to 90,000 cells/min, from 10,000 to 80,000cells/min, from 10,000 to 75,000 cells/min, from 10,000 to 70,000cells/min, from 10,000 to 60,000 cells/min, from 10,000 to 50,000cells/min, from 10,000 to 40,000 cells/min, from 10,000 to 30,000cells/min, from 10,000 to 20,000 cells/min, from 10,000 to 15,000cells/min, from 40,000 to 100,000 cells/min. In some exemplaryembodiments, any of the above-described systems and methods can furthercomprise sorting at least 10,000 cells/min, at least 15,000 cells/min,at least 20,000 cells/min, at least 25,000 cells/min, at least 30,000cells/min, at least 35,000 cells/min, at least 40,000 cells/min, atleast 45,000 cells/min, at least 50,000 cells/min, at least 75,000cells/min, at least 100,000 cells/min, at least 125,000 cells/min, atleast 150,000 cells/min, at least 200,000 cells/min, at least 250,000cells/min, at least 300,000 cells/min.

FIG. 2 illustrates a non-limiting example of a microfluidic device 200can comprising three outlets (230 a, 230 b, 230 c). The microfluidicdevice 200 can comprise, for example, a top outlet 230 a, a centraloutlet 230 b, and a bottom outlet 230 c. As a result, the cellscollected at the top 230 a and bottom 230 b outlets can have differentexpressions for a target adhesion molecule.

The disclosed microfluidic devices may comprise an expansion region anda plurality of hydrodynamically balanced outlets. FIG. 2 illustrates anexemplary and non-limiting three-outlet microfluidic device 200comprising an expansion region 240 and three hydrodynamically balancedoutlets (230 a, 230 b, 230 c). The hydrodynamically balanced outlets(230 a, 230 b, 230 c) can each independently comprise a flowapportionment region 260, a flow balancing region 270, and a collectionpoint 280. The expansion region 240 can comprise a compressivesurface-free portion of the microfluidic channel 250 comprising theplurality of compressive surface. The expansion region 240 can be influid communication with the flow apportionment regions 260 of theoutlets (230 a, 230 b, 230 c). The expansion region 240 and flowapportionment regions 260 can have an added benefit of evenly dividingchannel flow amongst the outlets (230 a, 230 b, 230 c). In someexemplary embodiments, at least one of the outlets can comprise a flowapportionment region that is larger or smaller than at least a flowapportionment region of another outlet. In a non-limiting example, asillustrated at FIG. 2 , the flow apportionment region of the bottomoutlet 230 c is larger than the flow apportionment region of the topoutlet 230 a and the bottom outlet 230 b.

The outlets (230 a, 230 b, 230 c) may also each independently comprise aflow balancing region 270 and a collection point 280. The flow balancingregion 270 can be downstream from and in fluid communication with theflow apportionment region 260. The collection point 280 can bedownstream from and in fluid communication with the flow balancingregion 270. The flow balancing region 270 can be designed so as toincrease the flow resistance in the outlet and prevent flow biasing fromuneven flow apportionment regions and external perturbations. Theoptimal architecture of the expansion region 240, flow apportionmentregions 260, and flow balancing regions 270 can be determined usingcomputational fluid dynamics to design a balanced channel flow egressacross all outlets, as illustrated at FIG. 2 . In a non-limitingexample, and as illustrated at FIG. 2 , the flow balancing regions 270can comprise a substantially serpentine architecture. As will beunderstood, the flow balancing region can comprise a variety of shapes,architectures, and lengths depending on the device design.

EXAMPLES

The present disclosure is also described and demonstrated by way of thefollowing examples. However, the use of these and other examplesanywhere in the specification is illustrative only and in no way limitsthe scope and meaning of the disclosure or of any exemplified term.Likewise, the disclosure is not limited to any particular embodimentsdescribed here. Indeed, many modifications and variations of thedisclosure may be apparent to those skilled in the art upon reading thisspecification, and such variations can be made without departing fromthe disclosure in spirit or in scope.

Example 1—Continuous Sorting of Cells Based on Differential P SelectinGlycoprotein Ligand Expression Using Molecular Adhesion

In an exemplary embodiment, the above-described systems and methods caninclude a microfluidic platform capable of high throughput separation ofcells by differences in molecular adhesion. The device can operate byflowing cells through a ridged microchannel such that the surfaces aredecorated with cell adhesion entity (e.g. an adhesion molecule)providing specificity to a cell surface receptor. As cells transientlyinteract with ligands at the ridge and wall interfaces, the net forcescan be altered to redirect the flowing cells toward one side of thechannel. Thus, cells with high expression of the target molecule can beconcentrated toward one side of the channel for collection. One uniqueaspect of this sorting design is the ridge gap spacing (e.g. acompression gap) that optimizes cell compressions to increase thesurface area for interaction between the ligand on cell surface andcoated receptor molecule, but without sufficient strain to undulyinfluence the cell trajectory by biomechanical properties such asstiffness and viscoelasticity. Thus, ridge compression can be used tooptimize adhesive interactions in a manner that cell stiffness does notinfluence the cell separation mechanism. As a result, receptor-specificcell separation can occur while maintaining a high flow rate andthroughput. By designing chips with multiple outlets, the sorting iscapable of fractionation of cells based upon the amount of receptorexpressed.

To demonstrate cell separation, lectin molecules were used, whichpreviously have been used for sorting cells though affinity columns.Since cell binding to lectins depends on factors like metabolic state,stage of cell division, and differentiation, the method is useful forapplications like isolating stem cells based on differentiation orhoming potential. Of these lectin-based sorting, P selectin and Pselectin glycoprotein ligand 1 (PSGL-1) sorting was chosen due to thepotential applications in understanding the role of PSGL in T cellimmune response and developing effective therapeutics. Therefore, inthis study, PSGL-1/P selectin, ligand-receptor binding is used to sorttarget cells.

Experimental Methods

Microfluidic Device Fabrication. Microfluidic sorting devices weredesigned in SolidWorks. The microfluidic devices with different gap sizewere fabricated by replica molding Polydimethylsiloxane (PDMS) on apermanent mold. The mold was made from SU-8 2007 using a two maskphotolithography process. The mold dimensions were characterized withprofilometry (Dektak 150 proflier) and verified with confocal microscopyimaging (Olympus LEXT).

Uncured PDMS was mixed in a 10:1 ratio of elastomer to curing agent(Sygard 184 elastomer kit), then poured onto the SU-8 molds to athickness of 1 cm and cured in an oven at 60° C. for six hours. Thecured PDMS layer was peeled off the mold, cut into chips, and inlet andoutlet holes were formed with a 1 mm biopsy punch. The PDMS device wastreated with oxygen plasma (Harrick plasma cleaner) for two minutes thenbonded to a glass microscope slide. The ridged microchannels weredesigned to have gap sizes of 9.3 μm for Jurkat cells and 10.3 μm forHL60 cells, which imposes a cellular strain of ˜15% on each cell type.The channel width was 560 μm and length was 3.8 mm with 25 skew ridgesequally spaced along the channel length. The ridges were 20 μm wide anddistance between two consecutive ridges was 70 μm. The ridges were atthe top of the microchannel and are inclined at 30 degrees. Three andfive outlet devices were used to study the resolution of separation.

Sample Preparation and Experimental Setup. Jurkat cells (CRL-1990) andHL60 (CCL 240) were used. Cells were cultured and maintained inRPMI-1640 medium with the addition of 10% FBS and 1%penicillin-streptomycin. Cells were incubated at 37° C. supplied with 5%carbon dioxide. Recombinant human P selectin was resuspended in PBS at aconcentration of 3 μg/mL. The assembled device was degassed in a vacuumchamber for 10 minutes, filled with P-selectin solution (3microliters/mL) by pipetting, and then incubated at room temperature.After three hours incubation, the device was washed with 1% bovine serumalbumin (BSA). Cells suspended in medium at 0.5×10⁶ or 1×10⁶ cells/mLwere flowed into the device at 0.045 and 0.1 mL/min using syringe pump.A high-speed camera (Phantom V7.3 149 Vision Research) and invertedmicroscope setup is used as described previously. Following collectionat outlets, cells are incubated at 37° C. with blocking solution for 15minutes and then incubated for 30 minutes with primary monoclonalantibodies and then, after a wash with PBS, incubated with fluorescentlylabeled secondary antibodies for 30 minutes at final concentrations of30 and 50 μg/mL respectively. Between primary and secondary antibodiesincubation and after secondary antibody incubation and flow analysis,cells were washed with PBS. To detect cell-surface PSGL-1, mouseantihuman PSGL-1 clone KPL-1 was used, followed by secondary antibodyPE-conjugated goat antimouse IgG. Solutions composed of primary andsecondary antibodies were preincubated for at least 1 h prior toincubation with cells. Cells were analyzed with flow cytometer.Fluorescent imaging was used to check the degree of detachment of Pselectin from cells after flow experiment. For this purpose, 1% FITC BSAwas used as a blocking agent. To measure the activation of cells,antibodies, anti CD69-FITC, and CD11b-APC were used according tomanufacturer's manual. Three sets of experiments were conducted for thestudy of cell activation. In the first experiment, incubated Jurkatcells were incubated with P selectin for 24 hours at 37° C. with 5%carbon dioxide. In the second experiment, Jurkat cells were incubatedfor two hours with P selectin coated PDMS surface, in which the PDMSsurface was first activated by incubating it with P selectin for threehours at room temperature, at 37° C. with 5% carbon dioxide. In thethird experiment, Jurkat cells were collected after sorting through theproposed device. Expressions of CD69 and CD11b were compared for all thethree experiments using flow cytometry.

Cell Stiffness Measurement with Atomic Force Microscopy. Atomic forcemicroscopy was used to measure the stiffness of cells. All cells weremeasured in suspended states after slight attachment to the surface. Tomeasure cells in suspended state, a monolayer of poly-L-lysine wasgrafted onto the glass slide substrate. This operation providedanchorage of the cell to the glass substrate while maintainingroundedness of morphology for cells and improved the cell stabilityduring the AFM measurements. The AFM experiment was carried outimmediately after the washing step and poly-L-lysine cell attachmenttreatment and all measurements were finished within two hours. A changein measured stiffness was not observed during the course of thesemeasurements. Measurements were conducted using a MFP-3D AFM attached toan inverted optical microscope. A silicon nitride cantilever with aspring constant measured to be 37.1 pN/nm and a spherical tip waspositioned above the center of individual cells before indentation. TheYoung's modulus is a function of loading force and loading rate. Theforce-indentation curve was obtained for each measurement at a 40%strain and then analyzed with a Hertzian model for a spherical tip fromwhich the Young's modulus values were calculated.

Fluid Flow Simulations. Finite element simulations of fluid flow wereperformed using COMSOL Multiphysics software. Simulations were performedfor channel width of 560 μm and ridge inclination angles of 30°. PDMSwas selected as the material of interfacing structure. The flow profilesin the channel were obtained by solving the Navier-Stokes equations forincompressible fluid using fluid-structure interaction physics. At theoutlet, the pressure was set to zero with no viscous stress on theboundary. Because of low Reynolds number of the fluid, it was assumedthat the suspended particles would follow the fluid streamlines.

Results and Discussion

The sorting device shown in FIG. 3A uses the flow of cells through amicrochannel decorated with diagonal ridges and selectin coated bottomglass surface on cells as they flow results in a net lateraldisplacement that distributes the cells at different y positions henceat different outlets based on the binding between P selectin on devicesurface and PSGL-1 on cell surface. The cells flowing in the devicewithout P selectin follow the fluid streamlines as no adhesion isobserved in this case. The unique aspect of this sorting design is theuse of optimized gap size height, which can include the distance betweenthe ridge and bottom of the channel, to lightly squeeze the cells whileflowing under the ridged part of the channel while offering a highsurface area for specific interaction between the cells and ligandmolecules coated on the ridges. FIG. 3B shows the trajectory of Jurkatcells flowing through the device with (black, 210) and without (blue,220) P selectin coating. Jurkat cells without P selectin coating tendedto follow trajectories to towards the side edges of the channel whereasJurkat cells with P selectin coating tended to follow a trajectorythrough the middle of the channel.

To understand the sorting of cells by expression of PSGL-1, the behaviorof two model leukocytes flowing in the device was examined, HL60 andJurkat cell lines. The gap size in each case was optimized so thatbiomechanical compression forces resulting from stiffness andviscoelasticity were minimized so as not to dominate the cell separationprocess. In these flow experiments, 9.3 μm gap size was used foradhesive sorting of Jurkat cells, which is larger than prior gap sizesreported for stiffness separation of Jurkat cells of 8 μm. Thus, the 9.3μm gap size is sufficient to lightly squeeze Jurkat cells, which have adiameter of 11 μm, but large enough that stiffness does not dominate thesorting. In the case of HL60 cells, a 10.3 μm gap size was used as thesecells are 12 μm in diameter. Specificity to PSGL-1 expression wasobtained through device functionalization by ligands found on surface ofHL60 and Jurkat cells and shows binding to P selectin coated surfaces.

To characterize the sorting of different cell types by the device, thesorted cells were examined after incubating them with primary andsecondary antibodies using protocol described in methods with flowcytometry. For both HL60 and Jurkat cells, cells were separated based onexpression of PSGL-1 ligand by using a single, ridged channel coatedwith P selectin. HL60 cells were flowed at flow rates of 0.045 and 0.1mL/min in the device with P selectin incubation and at 0.045 mL/minwithout P selectin incubation. The flow cytometry data for outlets forthe three different cases are shown in FIG. 4A. Adhesion dependence ofoutputs faced an upper limit of flow rate in which sorting of PSGL-1positive cells at high flow rates was diminished, as shown in FIG. 4A,which shows flow cytometer data for HL60 cells collected at differentoutlets, including the peak shift in their mean fluorescent values. FIG.4B shows the fluorescent mean values of collected sample at differentoutlets for 0.045 mL/min flow rate with P selectin incubation. A two-wayANOVA and Tukey tests were performed on the collected data to show asignificant difference in fluorescent mean values of three outlets. Theutilized flow rate of 0.045 mL/min was substantially higher than thatused in other microfluidic label-free adhesion based sorting. 3.8 and3.2-fold enrichment of PSGL-1 positive and negative HL60 cellsrespectively was demonstrated, as shown in FIG. 4C. Fluorescentmicroscopy was also used to validate outlet characterization of thedevice, as shown in FIG. 4D. The PSGL-1++ outlet in FIG. 4D shows morecells with secondary antibody attached to them, hence showing morePSGL-1 expression.

FIG. 5A analyzes the trajectory of HL60 cells with and without Pselectin functionalization and at increased flow rates. In FIG. 5B, thedata from trajectories was extracted to determine the lateraldisplacement of cell flows between two ridges, defined as Δy/ridge. Thetrajectories for uncoated devices showed displacement only in thenegative y direction; whereas, the P-selectin coated device showeddisplacement with a range of values, either positive, negative, or inthe middle of the device.

Analysis of trajectories of cells at high flow rate of 0.1 mL/min for Pselectin coated device in FIGS. 5D-5E shows that 40% of the cells aredisplaced in negative y direction with 7.3 μm and 60% in positive ydirection with 5.3 m Δy/ridge and at much higher velocities as comparedto 0.045 mL/min flow rate. This lack of enrichment at high flow rates(FIG. 4A, right) indicates hydrodynamic forces dominate adhesive forces,in part due to larger hydrodynamic forces and in part to insufficienttime for cells to form adhesions with the coated surface. Therefore, lowenrichment of cells based on adhesion was observed at a flow rate of 0.1mL/min.

The working mechanism of the device is explained in FIG. 6A. As cellsflow under the ridge, they bind to P-selectin and when leaving theridges cells with more ligand expressed on their surface resist thesecondary drag force and stay adhered to the bottom of the channel andflow in a negative y direction (red arrow). On the other hand, cellswith less ligand expression detach and pull away from the surface andenter the streamline in a positive y direction (green arrow). Cellsflowing through the device without P selectin coated ridges move withthe fluid flow streamlines. The trajectories of Jurkat cells arecompared with COMSOL streamline at height equals to half of the gap sizein xy-plane in FIGS. 6B-6D. Cells closely follow the simulatedstreamline in the case of a device with no P selectin. Since cells inthe stream are located near the bottom channel wall with weak elasticforce and no adhesion force, they are transported by the circulatingflow created by the ridges in the negative transverse direction, asshown in FIG. 6B. In the case of less PSGL-1 on the cell surface (FIG.6C), as a cell leaves the ridge, it is pulled off and detached from thesurface and follows the streamlines that moves up in y direction. On theother hand, a cell with more PSGL-1 (FIG. 6D) attaches to the surfaceonce it enters the ridge and rolls nearly straight on

In previous studies, gap sizes ranging from 4 to 12 μm were tested forsorting Jurkat cells by stiffness and found that an 8 μm gap size gaveoptimal sorting based on biophysical differences with minimal occlusion.On the basis of these conclusions, gap sizes of 9 and 14 μm were testedto evaluate sensitivity to adhesion differences without sensitivity tostiffness differences. As can be seen in FIGS. 7A and 7B, increasing thegap size decreases the enrichment factor because of a decrease in cellstrain, which leads to decrease in surface area for interaction betweencell surface and coated device. After cell separation, the stiffness ofthe cells collected at the three outlets was also examined, as shown inFIG. 7C. A lack of significant difference in stiffness indicates thatstiffness was not a determining factor in cell sorting. The measuredcell size distribution, representing the average plus and minus thestandard deviation of HL60 cells, was between 10.0 and 15.4 m (FIG. 7E).From this distribution and the ridge gap dimension, approximately 8% ofcells were exposed to <3% cell strain and ˜92.7% of cells were exposedto cell strain from 3% to 33%. A threshold of >40% cell strain isrequired for cell stiffness to dominate the sorting mechanism in aridged microchannel. While large cell heterogeneity in size or stiffnessmay indeed impact adhesion separation, for the conditions of this study,cell stiffness did not vary at the sorting outlets. It was anticipatedthat if cell heterogeneity significantly exceeded that studied here,i.e. if cell size variation exceeded 25%, then decreased accuracy ofadhesion dependence is expected. Fluorescent imaging of the device (FIG.7F) after the flow experiment verified that P selectin was notsignificantly removed after flow experiments and remained intactthroughout the flow experiment. The device after the flow experiment(middle image of FIG. 7F) showed no fluorescence and hence P selectin isintact after flow experiment.

FIG. 8A shows the flow cytometer data for Jurkat cells collected atdifferent outlets showing a peak shift in their mean fluorescent values.FIG. 8B shows mean fluorescent intensity at each outlet. 26-fold and4.4-fold enrichment of PSGL-1 positive and negative Jurkat cellsrespectively was demonstrated, as shown in FIG. 8C. Fractionation of asingle cell type based on the expression of a ligand at high flow ratesand with significant population enrichment was demonstrated, as shown inFIG. 8D. One potential application for fractionation of T cells issorting based on the density of specific chimeric antigen receptors(CAR). Potential targets for CAR-based therapies are cell surfaceantigens expressed at higher densities on cancer cells. Generally, thismay lead to severe adverse effects due to the recognition of minimal Agexpression outside the target tumor. The microfluidic sorting device cantherefore be used to determine threshold Ag densities for CAR-basedtherapies in order to avoid off target tumor toxicity.

A concern of adhesion-based isolation methods is the modification andactivation of the target cells after isolation by long-term binding(greater than 1 hour). Beads used for binding and pull-down isolationcan activate cells. Studies have also indicated that engagement ofselectin ligands on leucocytes directly transduces signals. For example,interactions of PSGL-1 with immobilized P-selectin rapidly inducetyrosine phosphorylation of multiple proteins. However, the present datashow that increase in tyrosine phosphorylation is observed at least twominutes after P selectin and PSGL-1 binding. The above-described systemsand methods provide another application where cell sorting is possiblewithout cell activation due to very short binding contact betweenreceptor and ligand. Three sets of experiments were conducted in orderto compare activation of cells due to short-term binding of PSGL-1 and Pselectin in the microfluidics sorting approach. To test activation ofthe sorted cells (data presented is for Jurkat cells), CD69 staining andCD11b were used to detect the activation of cells as classical markers.The results in FIG. 9 show a slight up regulation of activation markersafter cells are incubated with P selectin for 24 hours but verysignificant change when removed from P selectin coated surface. In FIG.9 , results are compared with cells incubated with P selectin for 24hours and with cells incubated with P selectin coated surface for 2hours. There is a significant up regulation of activation markers aftercells removed from P selectin coated surface. There was no change inexpression of activation markers in case of cells collected aftersorting. Also, no change in shape was observed. Taken together, theseresults indicate sorting produces no cell activation due to very shortbinding time (<10 s) between P selectin and PSGL-1 while cells areflowing through the shear in the device and the association anddissociation were sufficiently gentle so as not activate cells. In thiswork, HL60 cell line were separated at a final concentration of 106cells/mL at a flow rate of 45 μL/min which resulted in a throughput ofapproximately 45,000 cells per minute. The high throughput is over twoorders of magnitude higher than previous microfluidic approaches usingasymmetric adhesive patches that require cell rolling to maintaincontact. The improvement results from the ability to flow cells at afaster velocity due to the forced contact at the narrow-gap ridges.

CONCLUSION

A microfluidic device capable of high throughput separation of cells bydifferences in specific cell adhesion has been demonstrated. Adhesion ismediated by specific adhesive events from cell surface PSGL-1 receptorsto surface immobilized selectins. The device displays sufficientsensitivity to adhesion so that differential receptor expression can bedistinguished and enriched. 26-fold and 3.8-fold enrichment of PSGL-1positive and 4.4-fold and 3.2-fold enrichment of PSGL-1 negative Jurkatand HL60 cells have been demonstrated, respectively. Enrichment ofPSGL-1 positive Jurkat cells to 3-fold using a five-outlet fractionationdevice has also been demonstrated. This is the first study that showsfractionation of single cell line based on the ligand it expresses athigh flow rates with significant population enrichment. Heterogeneity incell stiffness or size show minimum effect on sorting at optimized gapsize. Because of the skewed ridge design, no clogging was observed andability to clear cells with less interrupted fluid flow. Further,applications using the device to sort desired cell phenotypes with highthroughput are possible to allow downstream purification and analysis.

The ligand-based sorting also has potential applications inunderstanding the role of PSGL-1 in T cell immune response anddeveloping effective therapeutics. It has been reported that PSGL-1 on Tcells dampens TCR signals, limits survival of effector T cells, andpromotes immune inhibitory receptor expression, thereby supportingestablishment of exhaustion in viral and tumor models. PSGL-1-deficiencyenhances T cell antitumor immunity to melanoma, promotes viral controland T cell survival is reported to be increased in PSGL-1 deficit Tcells after chronic virus infection. The above-described systems andmethods also offer direct measurement of transient nature interactionbetween important physiological ligands and interacting cells. It can beused as a tool to monitor stem cell differentiation. It also haspotential applications in measuring degree of changes in adhesionsignature of cancer cells, hence better therapeutics. Unlike MACS andpanning, the separation can fractionate by the amount of surface antigenon a cell and also eliminate the limitation posed by the choice ofantibodies is limited within a pool of commercially availableantibodies, which in turn limits the separation targets to those cellswith specific markers.

It will be clear to a person skilled in the art that features describedin relation to any of the embodiments described above can be applicableinterchangeably between the different embodiments. The embodimentsdescribed above are examples to illustrate various features of theinvention.

The reader's attention is directed to all papers and documents which arefiled concurrently with or previous to this specification in connectionwith this application and which are open to public inspection with thisspecification, and the contents of all such papers and documents areincorporated herein by reference.

Numerous characteristics and advantages have been set forth in theforegoing description, together with details of structure and function.While the invention has been disclosed in several forms, it will beapparent to those skilled in the art that many modifications, additions,and deletions, especially in matters of shape, size, and arrangement ofparts, can be made therein without departing from the spirit and scopeof the invention and its equivalents as set forth in the followingclaims. Therefore, other modifications or embodiments as may besuggested by the teachings herein are particularly reserved as they fallwithin the breadth and scope of the claims here appended.

We claim:
 1. A microchannel for processing cells comprising: an inletconfigured to deliver at least one of a first portion of the cells and asecond portion of the cells in the microchannel; ridges, each ridgecomprising: a compressive surface configured to compress the cells; anda cell adhesion entity; and an outlet configured to remove at least oneof the first portion of the cells and the second portion of the cellsfrom the microchannel; wherein: each ridge is oriented at an angle offrom 25 degrees to 70 degrees relative to a center axis of themicrochannel; the cell adhesion entity is configured such that the firstportion of the cells has a first adhesion property relative to the celladhesion entity to follow a first trajectory through the microchannel;the cell adhesion entity is further configured such that the secondportion of the cells has a second adhesion property relative to the celladhesion entity to follow a second trajectory through the microchannel;and the first trajectory is different from the second trajectory.
 2. Themicrochannel of claim 1 further comprising a first wall and a secondwall; wherein the first wall is substantially parallel to the secondwall; and wherein each ridge protrudes from the first wall in adirection normal to the first wall and defines a compression gap betweenthe compressive surface and the second wall.
 3. The microchannel ofclaim 2, wherein the compressive surface of each ridge is substantiallyparallel to each of the first wall and the second wall.
 4. Themicrochannel of claim 2, wherein the compression gap has a height offrom 75% to 95% an average diameter of the cells.
 5. The microchannel ofclaim 1 further comprising a flow space disposed between an adjacentpair of ridges along the center axis of the microchannel; wherein theflow space has a width, along the center axis, of from 50 micrometers to500 micrometers.
 6. The microchannel of claim 5, wherein the flow spacewidth is from 100 micrometers to 300 micrometers.
 7. The microchannel ofclaim 1 further comprising one or more sheath inlets; wherein the inletis a cell focusing inlet; and wherein the one or more sheath inletssurround the cell focusing inlet.
 8. The microchannel of claim 1,wherein the first adhesion property is defined by a cell surfacereceptor of the cells; and wherein the cell adhesion entity is furtherconfigured to temporarily bind to the cell surface receptor of thecells.
 9. The microchannel of claim 1, wherein the first trajectory isdetermined based on the first adhesion property; and wherein the secondtrajectory is determined based on the second adhesion property.
 10. Themicrochannel of claim 1 further comprising an additional outlet; whereinthe outlet is configured to remove the first portion from themicrochannel; and wherein the additional outlet is configured to removethe second portion from the microchannel.
 11. The microchannel of claim1, wherein the angle at which the ridges are oriented varies along thecenter axis of the microchannel.
 12. The microchannel of claim 1,wherein the ridges have a thickness that varies along the center axis ofthe microchannel.
 13. The microchannel of claim 12, wherein each ridgehas a thickness from 2 micrometers to 30 micrometers.
 14. Themicrochannel of claim 1, wherein at least a portion of the cell adhesionentity is positioned on the compressive surface of each ridge.
 15. Themicrochannel of claim 1, wherein the cell adhesion entity is positionedonly on the compressive surface of each ridge.
 16. The microchannel ofclaim 1, wherein the microchannel comprises more than one type of celladhesion entity; and wherein different types of the cell adhesion entityare positioned on different ones of the compressive surfaces of theridges.
 17. The microchannel of claim 1, wherein the microchannelcomprises from 5 to 100 compressive surfaces.
 18. The microchannel ofclaim 1, wherein the microchannel comprises from 5 to 50 compressivesurfaces.
 19. The microchannel of claim 1, wherein the microchannelcomprises from 7 to 40 compressive surfaces.
 20. The microchannel ofclaim 1, wherein each ridge is oriented at an angle of from 30 degreesto 60 degrees relative to the center axis of the microchannel.